The major components of lipids in the serum and plasma are cholesterols, triglycerides, phospholipids and the like. These blood lipids circulate in the blood as lipoproteins in which the lipids are bound to apoproteins. The lipoproteins can be classified based on their densities into chylomicron (hereinafter referred to as CM), very low density lipoprotein (hereinafter referred to as VLDL), intermediate density lipoprotein (hereinafter referred to as IDL), low density lipoprotein (hereinafter referred to as LDL), high density lipoprotein (hereinafter referred to as HDL) and the like. Among these lipoproteins, HDL has an action to transport excess cholesterol deposited in tissues to the liver, and an antiarteriosclerotic action. LDL is a major carrier in transportation of cholesterol from the liver to tissues, and an increase in LDL is considered to have a strong association with occurrence of arteriosclerosis. Thus, cholesterol in LDL (hereinafter referred to as LDL-C) is considered to be a risk factor for arteriosclerosis, ischemic heart disease (coronary artery disease)and the like, and the LDL-C content has been regarded as an important index to be known for diagnosis, therapy and prophylaxis of these diseases. On the other hand, there are also many cases where ischemic heart disease or the like has developed even with a blood LDL-C level within the normal range. Thus, recent interest has focused on changes in the quality of LDL particles, and constituents other than cholesterol.
LDL containing a large amount of triglycerides (hereinafter referred to as TG-rich LDL) are lipoprotein having properties different from those of normal LDL, which contain a large amount of cholesterols. TG-rich LDL is found in a large amount in the blood of patients with liver disease, and its blood level increases as the liver disease progresses. It is reported that TG-rich LDL accounts for a large part of lipoproteins present in the blood at the end stage of hepatic disease. TG-rich LDL causes formation of macrophage foam cells. It is reported, for example, that the rate of formation of macrophage foam cells by TG-rich LDL is directly proportional to the serum level of malondialdehyde-modified LDL, which is a type of oxidized LDL, and that, although peroxidized triglycerides can be hardly detected in the blood of healthy individuals, peroxidized triglycerides are remarkably increased in the blood of patients with liver disease. Thus, triglycerides in LDL are thought to have strong association with oxidized LDL. That is, the amount of triglycerides in LDL can be considered to be an important index associated with liver disease, and various arteriosclerosis, coronary artery disease and the like that are associated with oxidized LDL.
Examples of the method for quantifying triglycerides in LDL (hereinafter referred to as LDL-TG) include the 2-step method by the combination of operations of fractionation and triglyceride quantification. Examples of the fractionation operation include methods using ultracentrifugation, electrophoresis, high-performance liquid chromatography (HPLC) and/or the like, and examples of the quantification method include a method in which the quantification is carried out using an automated analyzer for clinical tests together with a reagent for measuring triglycerides. LDL-TG can be quantified by the combination of these, but, since the quantification is carried out in 2 steps, that is, the pretreatment step in which LDL is completely separated from the lipoproteins other than LDL and the step in which the measurement is carried out, the operations are laborious and time-consuming. Moreover, depending on the separation method, recovery of the separated sample itself may be difficult, or quantitative recovery of the sample may be difficult. Even in a method that allows quantitative recovery, the operation may require a high level of skill or a special apparatus. Thus, these methods are costly, and unlikely to be commonly employed from the viewpoint of simplicity and economy.
Known examples of methods that can solve these problems and allow measurement using an automated analyzer or the like without requiring a fractionation operation include a method in which triglycerides in all non-LDL lipoproteins are removed in the first step, and triglycerides in the remaining LDL are measured in the second step (Patent Document 1), and a method in which (free glycerol and)triglycerides in HDL are removed in the first step, and triglycerides in only LDL are measured in the second step (Patent Document 2).
However, in these methods, triglycerides in VLDL and triglycerides in CM cannot be completely removed in the first step in cases where the sample contains a large amount of these triglycerides, and the reaction proceeds also in the second step in such cases, resulting in a positive influence. Moreover, in these methods, insufficient suppression of triglycerides in VLDL, whose reaction needs to be suppressed in the second step, may cause partial promotion of the reaction, resulting in a positive influence. Although positive correlations can be actually seen when these methods are compared with the definitive method, ultracentrifugation, using a number of samples, the above methods can be seen to have serious problems in specificity and accuracy to samples, such as the existence of samples showing largely different values, and a large variation.